Medical and research laboratories study tissues and cells using a microscope. Researchers and medical professionals typically require a visual contrast between organelles within a cell, and between the cell and the extra-cellular matrix to study the tissues and cells. A special optical method such as a phase contrast can partially remedy this situation. A method of choice is to process specimens with a staining method such as applying dyes or colors to tissues to increase contrast. A widely used histological stain is hematoxylin and eosin (H&E). For example, cell nuclei can be stained black and/or blue in color by the hematoxylin component stains, and cell cytoplasm and most connective tissue fibers can be stained in various intensities and/or shades of red, pink, and/or orange by eosin stains. The contrasting method can reveal more than merely revealing the cell and organelles in tissue. Furthermore, with the use of antibodies and nucleic acid probes, it is also possible to detect some specific antigens and nucleic acid sequences giving information on the physiological condition of cell and tissues that can be used for medical diagnostic and research. The procedures to achieve these results are long and tedious and, as a result, expensive. Existing tissue stainers and immuno-stainers are large, expensive, and limited to few applications.
The reason for the expense is due in part to the need for tissue samples to be removed from a patient and processed by several organizations for histological examination. The characteristics of a tissue sample often provide important information regarding the health of an individual, but the characteristics are impossible to determine without assistance from multiple labs, personnel and diagnosticians due to the complexity required for staining the tissue samples.
The process typically begins by removing a tissue sample from a living organism, fixing the sample, embedding the fixed tissue in a material such as paraffin, and slicing the embedded fixed tissues into very thin sections. This procedure produces a histological specimen.
The histological specimens prepared according to either the paraffin-embedding method can be analyzed in a variety of ways, such as staining the sample to identify nucleic acids, or probing the sample with detectably labeled antibodies.
Biological analysis using thin sections of embedded fixed tissue frequently applies to the diagnosis and prognosis of diseases and conditions such as cancer. For instance, a biopsy may be performed to determine whether a core or fragment of tissue removed from the patient is cancerous. Thus, a histological specimen is prepared from the tissue. The specimen is analyzed microscopically to determine whether the tissue exhibits the hallmarks of neoplasia or cancer.
It is known that tissue microarrays can be configured by combining hundreds of tissue samples in a single paraffin block to enable multiple tissue samples to be analyzed simultaneously. (Kononen, J, et al., Nature Medicine 4:767-768 (1998)). Thin sections from the tissue can later be analyzed using a variety of techniques, including DNA and RNA in situ hybridization and immunohistochemistry. (Bubendorf, L, et al., Cancer Res. 59:803-806 (1999)). Immunohistochemical markers or labels are often used in histology for identifying certain characteristics of a cell, for example, whether the cell is undergoing mitosis or expression of a certain antigen.
A known issue with the variety of techniques available to tissue samples is that many small labs and doctor's offices and hospitals lack the facilities to perform the analysis due to the complexity of the current techniques.
Typically, a doctor's office or laboratory can take biopsies and transfer them to laboratories for setting in paraffin or other fixative. After tissues are set in a fixative, transfer of the tissues to other specialized analysis locations takes place due to the complexity of preparing a slide for proper analysis. Fixed tissues can be stored within a vapor phase liquid nitrogen freezer system to maintain tissue. For example, deparafinization requires paraffin removal from the section of tissue with xylene. If the tissues are to be stained with an aqueous solution, then the slides must be rehydrated in graded ethanol baths. Typically, the approach is to gently agitate the slides by repeated immersion −20× in each bath including xylene for 2 minutes, 100% EtOH (×2), 95% EtOH (×1), 80% EtOH (×1), and H2O (×1).
One common stain for formalin fixed paraffin tissues is Hematoxylin and Eosin (H&E). Hematoxylin stains negatively charged nucleic acids blue. The eosin stains proteins pink. The hematoxylin or the eosin can also be used by themselves in more dilute form as counterstains for immunoperoxidase staining. Such staining requires dilution of the stain 1:4 with H2O or EtOH, respectively. Slides to be stained must be washed in ethanol, and then washed with Hematoxylin, 2 minutes (×1), Running water (×1), Acid alcohol (×1), H2O (×1), Ammonia solution (×1), Running water 5 minutes (×1), 80% EtOH (×1), Eosin 15 seconds, 95% EtOH (×2), 100% EtOH (×2).
A stain appropriate for auto-staining is the “Wright Giemsa” stain which requires heat fixation by warming a slide. Next, the slide can be exposed to alcohol, 30% to 80%. The stain remains on the slide for 30 to 60 seconds, followed by a running water rinse, drying and covering with oil or slide adhesive.
Another stain used for slides includes a benzidine stain that is a specialized stain configured to identify erythroid cells. To stain such a slide requires a methanol bath, 10-15 seconds followed by benzidine for 5 minutes, then peroxide, dionized water for 2.5 minutes, hematoxylin stain for 1.5 minutes, and a water rinse.
The above sampling of staining protocols and chemicals provides ample reason why such complex procedures are performed in specialized laboratories. Given the different protocols for preparing slides for stains, the toxic chemicals used such as benzidine, typical doctor's offices and local laboratories have limited resources for known auto-staining systems that are capable of a plurality of auto-stainings. Typically, such local operations will invest in simple stain auto-stain systems.
Accordingly there is a need for an efficient and reduced complexity system and method for tissue staining and analysis that enables laboratory analysis and staining of tissue samples.